Hi Guys,
I've recently started doing ChIP-qPCR and have questions regarding analysis of data obtained from qPCR.
My experimental design: I'm transfecting cells with a luciferase reporter plasmid containing a promoter that is stimulated by my transcription factor (TF) of interest. When I perform the luciferase assay I include a DNA-binding mutant of the TF that is unable to stimulate any luciferase expression. I need to show that the mutant version is indeed unable to to bind DNA resulting in no activation of luciferase expression. For this purpose we chose to do ChIP-qPCR. So when I setup for ChIP I've got one plate of cells with TF of interest + reporter and another plate of cells with mutant TF + reporter.
During ChIP I include a no-antibody (Mock) control to check specificity of antibody. ChIP-qPCR results are as below:
Cells transfected with TF of interest:
Ct input (10%) = 24.42246151
Ct with antibody = 28.97134145
Ct no antibody = 32.08292007
Cells transfected with mutant TF:
Ct input (10%) = 23.01083565
Ct with antibody = 29.89976883
Ct no antibody = 32.16822243
I use the percent input method [100*2^(Adjusted input - Ct (IP)] to calculate enrichment (pardon me if this is not the correct term to use here). I got the formula from here: http://www.lifetechnologies.com/au/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html
With this method I get the following values:
% input TF of interest (with antibody) = 0.42721913
% input TF of interest (no antibody) = 0.049427904
% input mutant TF (with antibody) = 0.084377077
% input mutant TF (no antibody) = 0.017512651
My question is how to present this data?
I also used primers for an unrelated region as everybody seems to 'normalize' their results to that. The values I got are below:
Cells transfected with TF of interest:
Ct input (10%) = 26.65428988
Ct with antibody = 30.54074732
Ct no antibody = 35.37199211
Cells transfected with mutant TF:
Ct input (10%) = 26.33532206
Ct with antibody = 31.8692112
Ct no antibody = 34.11607869
% input TF of interest (with antibody) = 0.67617597
% input TF of interest (no antibody) = 0.023752544
% input mutant TF (with antibody) = 0.215840718
% input mutant TF (no antibody) = 0.045473551
I was assuming that cells transfected with TF of interest and those with mutant TF will show similar % input for the unrelated region but that clearly is not the case and it seems like the TF of interest perhaps does bind to this region, although the Ct values are high (since they are in the 30s?)..
So if I can get any help in analysing this data and some suggestion on how to present it that would be great. I need to find a way that'll convince the reader that the wildtype (TF of interest) does bind and the mutant TF has reduced capability.
Thanks heaps!!
Jerry
That was surprising for me too. The unrelated region belongs to a gene whose expression should be unaffected by my TF of interest (in theory). I used it because somebody in my lab had already used this region for their RT-PCR analysis.
I am new to this field, Can anybody please explain what is difference between Ct (INPUT) 10% and Ct no antibody?
Hi Nguyen,
the number 6.644 relates to the number of additional cycles which are needed to detect a 1:100 diluted sample at the threshold during qPCR in comparison to the undiluted sample (if an PCR efficiency of 100% is assumed) => 2^6.644 =100
For your dilution (1:20) you would need to substract 4.322
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